1. Field of the Invention
The invention relates to the diagnosis of herpes simplex virus type 2 (HSV-2) antibodies in samples, especially serum samples, taken from patients suspected of being infected by this virus. The invention provides multiply-displayed peptide structures useful for this purpose and a method and kit for diagnosis using these peptide structures and also monomeric peptides useful for preparation of the multiply-displayed peptides.
2. Description of the Related Art
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are two closely related viruses that infect humans. Most people over age 15 have antibodies to HSV-1 or -2 indicating that they have been infected with and harbour these viruses. Both viruses produce orogenital lesions and can also infect the eye, skin and nervous system. HSV-1 is mainly responsible for oral lesions while HSV-2 is mainly responsible for genital lesions. Following primary infection, the virus can enter a latent state in neurological tissue from which it can periodically reactivate to produce recurrent infections. Reactivations are frequently asymptomatic so that virus can be shed and transmitted in the absence of overt clinical lesions.
The need for a simple and inexpensive serodiagnostic test capable of distinguishing HSV-1 from HSV-2 antibodies was recognised at the WHO recent conference (1996). Serotyping of HSV will be important in trials of HSV vaccines in order to establish the immunological status of individuals before vaccination. It is hoped to prevent neonatal herpes by screening pregnant women and their sexual partners for asymptomatic HSV-2 infections and, where appropriate, recommending precautions designed to prevent transmission.
Progress towards an HSV-2-specific serodiagnostic reagent was facilitated by the identification more than 10 years ago of an HSV-2 glycoprotein, designated 94K or gG2, (Marsden et al, 1978, 1984; Roizman et al., 1984). Subsequently, the HSV-1 counterpart, gG1 was identified. Determination and comparison of the DNA sequence of the genes encoding gG1 and gG2 showed the two proteins to have diverged considerably. The existence of serotype-specific epitopes on gG1 and gG2 (Ackerman et al., 1986; Frame et al., 1986; Marsden et al., 1984, Roizman et al., 1984) raised the possibility of the use of gG1 and gG2 as serotype-specific diagnostic reagents.
Various serological tests for HSV-2 specific antibodies have been developed using gG2. These tests include ELISA, immunodot and western blotting assays, and serum blocking assays using serotype-specific monoclonal antibodies. For these purposes, gG2 has been obtained from a variety of sources including HSV-2 infected mammalian cells, transformed mammalian cells, insect cells infected with recombinant baculoviruses and Escherichia coli using several expression vectors. E. coli has also been used to express fragments of gG2 containing type-specific regions, and slightly truncated, almost full length fragments of gG2 fused to the maltose binding protein. Antigens from these sources have been used either as crude extracts or following purification by a variety of procedures including ion-exchange, monoclonal antibody-affinity and lectin-affinity chromatography.
Amongst the serological tests using gG2, western blotting is considered the most reliable (WHO conference, Copenhagen, 1995). However, this method is cumbersome, relatively expensive and not suitable for general screening purposes in other than well-equipped diagnostic laboratories. There is a need for a simple, cheap and reliable serodiagnostic test which can be used as an ELISA (Enzyme-Linked ImmunoSorbent Assay) in microtitre plates.